The present invention provides recombinant methods and materials for producing polyketides by recombinant DNA technology. The invention relates to the fields of agriculture, animal husbandry, chemistry, medicinal chemistry, medicine, molecular biology, pharmacology, and veterinary technology.
Oleandomycin (compound (1) of FIG. 1) is a member of the macrolide class of antibiotics. Macrolides are a large family of polyketide natural products which include erythromycin, spiramycin, FK506, and avermectin (see Katz et al., Polyketide synthesis: Prospects for hybrid antibiotics, Ann. Rev. Microbiol. 47: 875-912, 1993; and Hopwood, Genetic contributions to understanding polyketide synthases, Chem. Rev. 97: 2465-2497,1997, each of which is incorporated herein by reference). The macrolactone core of oleandomycin, 8,8a-deoxyoleandolide (compound (2) of FIG. 1), like those of other macrolides, is synthesized by a modular polyketide synthase (PKS; see FIG. 1 and Swan et al., Characterisation of a Streptomyces antibioticus gene encoding a type I polyketide synthase which has an unusual coding sequence, Molec. Gen. Genet. 242: 358-362, 1994, and U.S. patent application Ser. No. 09/428,517, filed Oct. 28, 1999, now U.S. Pat. No. 6,251,636 which issued on Jun. 26, 2001, each of which is incorporated herein by reference). 8,8a-deoxyoleandolide is structurally identical to the macrolactone precursor of erythromycin, 6-deoxyerythronolide B (6-dEB, see compound (3) of FIG. 2), with the exception of a C-13 methyl instead of the C-13 ethyl group of 6-dEB. Thus, 6-dEB is derived from condensations between a propionate starter unit and six methylmalonate extender units, and 8,8a-deoxyoleandolide has an acetate starter unit.
The study of oleandomycin biosynthesis has been progressive over the past decade, due largely to the identification and sequencing of several biosynthetic and related genes by Salas and coworkers. Analysis of these gene sequences has revealed enzymes putatively involved in synthesis and attachment of the two deoxysugars, regulatory and antibiotic resistance genes, and a P-450 monooxygenase (see Rodriguez et al., A cytochrome P450-like gene possibly involved in oleandomycin biosynthesis by Streptomyces antibioticus, FEMS Microbiol. Lett. 127: 117-120, 1995; Olano et al., Analysis of a Streptomyces antibioticus chromosomal region involved in oleandomycin biosynthesis, which encodes two glycosyltransferases responsible for glycosylation of the macrolactone ring, Mol. Gen. Genet. 259: 299-308, 1998; and Quiros et al., Two glycosyltransferases and a glycosidase are involved in oleandomycin modification during its biosynthesis by Streptomyces antibioticus, Mol. Microbiol. 28: 1177-85, 1998, each of which is incorporated herein by reference). Thus, a single open reading frame (ORF) encoding a polypeptide subunit of a type I polyketide synthase was identified and, based on comparison to 6-deoxyerythronolide B synthase (DEBS), was hypothesized to encode the last two modules of the oleandomycin PKS (OlePKS; see Swan et al., supra). The cloning, characterization, and sequence determination of the other genes encoding the proteins of the OlePKS are described in PCT patent publication No. 00/026,349, incorporated herein by reference.
There remains, however, a need for additional methods and reagents to produce 8,8a-deoxyoleandolide, oleandomycin, and related compounds in heterologous host cells. The present invention meets these and other needs.
The gene cluster encoding the deoxyoleandolide polyketide synthase (OlePKS) was isolated from the oleandomycin producing strain Streptomyces antibioticus. Sequencing of the first two genes encoding OlePKS, together with the previously identified third gene revealed an overall genetic and protein architecture similar to that of the erythromycin gene cluster encoding the 6- deoxyerthronolide B synthase (DEBS) from Saccharopolyspora erythraea. When the entire OlePKS (10,487 amino acids) was expressed in the heterologous host Streptomyces lividans, it produced 8,8a-deoxyoleandolide (compound (2) of FIG. 1), the aglycone precursor of oleandomycin. The P450 monooxygenase, OleP, involved in oleandomycin biosynthesis was co-expressed with DEBS in S. lividans. The production of 8,8a-dihydroxy-6-deoxyerythonolide B (compound (6) of FIG. 2) and other derivatives demonstrates that OleP is involved in the epoxidation pathway of oleandomycin biosynthesis. Further, a method of producing 8,8a-dihydroxy-6-deoxyerythonolide B or 8,8a-dihydroxyoleandolide comprising the heterologous expression of DEBS or Ole PKS, respectively, with OleP in S. lividans was developed. This heterologous expression system provides a means to produce these compounds, which can directly or after further modification be hydroxylated and glycosylated to provide useful macrolide antibiotics.
Thus, in one embodiment, the present invention provides a method for introducing one or more hydroxyl groups or an epoxide into a polyketide, which method comprises expressing a recombinant gene encoding a P450 monoxygenase in a host cell. In one embodiment, the P450 monooxygenase is not naturally expressed by the host cell. In another embodiment, neither the P450 monooxygenase nor the polyketide is naturally expressed by the host cell. In a preferred embodiment, the host cell is a Streptomyces host cell. In another preferred embodiment, the P450 monooxygenase is OleP. In another preferred embodiment, the polyketide synthase is 6-deoxyerythronolide B synthase or 8,8a-deoxyoleandolide synthase.